The invention relates to screening compounds for antimicrobial activity, and, more particularly, to using bacterial proteins in vitro to detect compounds that interfere with cell division.
Antimicrobials are developed on the principle of selective toxicity. That is to say, antimicrobials, while toxic to the microorganism, must not be toxic to the patient. The selective toxicity of these drugs is usually relative, rather than an absolute. This means simply that most drugs are given to patients in concentrations that are tolerated by the patient, but are lethal or damaging to the microorganism; higher doses would be toxic to the patient and are avoided.
Selective toxicity is often a reflection of the presence of specific receptors present on the microorganism, but lacking in the host system. Other means to achieve selective toxicity commonly rely on the inhibition of biochemical events essential to the microorganism but not the host. As the physiology, structure, and biochemical systems of infectious agents and their hosts are usually quite different, antimicrobial development often relies on these differences.
Although the mechanisms of action of many antimicrobials are not well understood, the five major categories of action include inhibition of cell wall synthesis, inhibition of cell membrane function, inhibition of protein synthesis, inhibition of nucleic acid synthesis, and interference with intermediary metabolism. (See e.g., W. K. Joklik et al., [eds.], Zinsser Microbiology, 18th ed., Appleton-Century-Crofts, Norwalk, Conn., [1984], p. 193). For example, penicillin, like all xcex2-lactam drugs, is a compound which selectively inhibits bacterial cell wall synthesis. The initial step in the mechanism of action of these xcex2-lactam drugs involves the binding of the drug to cell receptors known as xe2x80x9cpenicillin-binding proteinsxe2x80x9d (xe2x80x9cPBPxe2x80x9d). There are from 3-6 PBPs, with molecular weights ranging from 4-12xc3x97105; some of these PBPs are transpeptidation enzymes. (Jawetz, Melnick and Adelberg""s Medical Microbiology, 19th ed, Appleton and Lange, Norwalk, Conn. [1991], p. 150). After binding to the PBP, the drug inhibits the transpeptidation reaction and synthesis of peptidoglycan in the organism""s cell wall material is blocked. This results in the eventual triggering of an autolytic cascade which leads to cell lysis.
Because of their relatively high concentration of peptidoglycan, gram-positive organisms tend to be much more susceptible to the effects of penicillin and other xcex2-lactams than gram-negative organisms. Importantly, because they affect cell wall synthesis, penicillin and the other xcex2-lactams are only effective against actively growing and dividing cultures. However, one of the benefits of these xcex2-lactam drugs is that animal cells do not have peptidoglycan; consequently, such drugs are remarkably non-toxic to humans and other animals.
Some organisms are naturally resistant to penicillin and the other xcex2-lactams due to their lack of PBPs, the inaccessibility of the PBPs due to the presence of permeability barriers, the failure of autolytic cascades to be activated following binding of the drug, or the lack of peptidoglycan in the cell wall (e.g., the mycoplasmas, L-forms, and metabolically inactive bacteria). Unfortunately, following years of use to treat various infections and diseases, penicillin resistance has become increasingly widespread in the microbial populations that were previously susceptible to the action of these drugs. Some microorganisms produce xcex2-lactamase, an enzyme which destroys the antimicrobial itself, while some microorganisms have undergone genetic changes which result in alterations to the PBPs, such that the drugs will no longer effectively bind to the receptors; still other organisms have evolved in a manner that prevents the lysis of cells to which the drug has bound. In this latter scenario, the drug has inhibited the growth of the cell, but it is not killed. In some circumstances this appears to contribute to the relapse of disease following premature discontinuation of treatment, as some of the cells remain viable and may begin growing once the antimicrobial is removed from their environment.
The development of tolerance and resistance to antimicrobials represents a significant threat to the ability to treat disease. Many factors have contributed to this increased observance of resistant strains, including over-use and/or inappropriate administration of antimicrobials, the capability of many organisms to exchange genetic material which confers resistance (i.e., R plasmids), and the relatively rapid mutation rate observed with many bacteria, which allows for selection of resistant organisms.
One well-documented example which highlights the problems with development of penicillin resistance involves Streptococcus pneumoniae, a gram-positive organism. Initially, the introduction of penicillin to treat S. pneumoniae resulted in a significant decrease in the mortality due to this organism. However, S. pneumoniae remains of great concern, as it is one of the agents most frequently associated with invasive infections; it is the most common cause of bacterial pneumonia and otitis media; it is the second most common cause of bacterial meningitis; and it is the third most common isolate from blood cultures. (J. F. Sessegolo et al., xe2x80x9cDistribution of Serotypes and Antimicrobial Resistance of Streptococcus pneumoniae Strains Isolated in Brazil From 1988 to 1992,xe2x80x9d J. Clin. Microbiol., 32:906-911 [1994]). Thus, the development of antimicrobial resistance in this organism is of great cause for concern.
The first report of pneumococci with decreased susceptibilities to penicillins occurred in 1967. Since this initial report out of Australia, additional strains with decreased susceptibilities have been reported worldwide. Additionally, resistance to penicillin alternatives, such as chloramphenicol, erythromycin, tetracycline clindamycin, rifampin, and sulfamethoxazole-trimethoprim has been reported, often in conjunction with penicillin resistance. Multiple-antimicrobial resistance in pneumococci was first reported in 1977. Since this initial report out of South Africa, multi-drug resistant strains have been reported in several countries, including Spain, Italy, France, Belgium, Hungary, Pakistan, Czechoslovakia, Canada, the United Kingdom, and the United States. (Sessegolo et al. supra, at 906).
In a survey conducted in Brazil, of 42 serotypes among 288 S. pneumoniae strains isolated during 1988-1992, Sessegolo et al. reported that decreased susceptibility to penicillin was detected in 26.7% of the strains. In addition, 35.9% of the strains were resistant to tetracycline, 29.2% were resistant to sulfamethoxazole-trimethoprim, 1.5% were resistant to rifampin, 0.80% were resistant to penicillin, and 0.50% were resistant to chloramphenicol. The penicillin-resistant strains were also found to be resistant to, or exhibited decreased susceptibility to cephalosporins. The resistance characteristics of these strains were also semi-quantitated, with intermediate resistances reported at 17.9% for penicillin, 8.7% for tetracycline, 6.7% for chloramphenicol, 6.1% for erythromycin, and 3.1% for rifampin.
Results obtained from patients in Rio de Janiero in 1981 and 1982, indicated that there was no penicillin resistance (relative or complete) in the pneumococcal isolates. However, during the period between 1988 to 1992, 19.4% of the strains from the same geographic population were relatively resistant, and 1.5% were completely resistant to penicillin. These results highlight the rapid spread of antimicrobial resistance.
Once an organism has developed resistance to a particular drug, it becomes important that an effective replacement drug be identified. If the organism develops resistance to this second drug, another replacement is needed. One example of the historical development of multiple drug resistance is gonorrhea. Prior to the 1930xe2x80x2s, treatment for this disease usually involved mechanical means, such as irrigation and use of urethral sounds in males. In the late 1930xe2x80x2s, sulfonamides were introduced and found to be effective in treating gonorrhea. After a few years, sulfonamide-resistant strains of N. gonorrhoeae were isolated. Fortunately, by this time, penicillin was available and found to be effective. However, by the 1970xe2x80x2s, many isolates of N. gonorrhoeae were found to be penicillin-resistant. This required the use of alternative drugs such as spectinomycin. It can be expected that this trend will continue, with the development of strains that are resistant to sulfonamides, penicillin, spectinomycin, and other antimicrobials.
Thus, there remains a need to develop new antimicrobials. Ideally, the antimicrobial should target the physiology of the microorganism and demonstrate selective toxicity. However, the targeting should, nonetheless, allow for antimicrobial action against a broad spectrum of organisms. Most importantly, the antimicrobial should serve as an effective replacement drug for multiple-drug resistant organisms.
The invention relates to screening compounds for antimicrobial activity, and, more particularly, to using bacterial proteins in vitro to detect compounds that interfere with cell division. The present invention contemplates the use of the zipA gene and gene product for screening compounds for potential antimicrobial activity. Unlike current screening approaches, the screening approach of the present invention does not require the use of bacterial cells.
The present invention contemplates the over-expression of recombinant ZipA protein that is functional, and yet free of contaminating protein typically associated with traditional biochemical isolation techniques. The expression of recombinant ZipA protein of the present invention relies on the construction of vectors (e.g., plasmids) containing the zipA gene and suitable hosts for protein expression. It is not intended that the present invention be limited by the expression system chosen for the expression of recombinant zipA. The present invention contemplates all forms and sources of expression systems (i.e., an expression vector/host cell combination).
In one embodiment, the present invention contemplates a method for screening compounds, comprising: a) providing: i) a test compound; ii) a first protein, said first protein encoded by an oligonucleotide comprising at least a portion of the zipA gene; iii) a second protein capable of binding to said first protein; and iv) means for detecting said binding; b) mixing said first and second proteins in the presence of said test compound; and c) detecting binding using said means for detecting binding.
The method may be performed using immobilized elements and the immobilization may be carried out using a variety of immobilization means (e.g., columns, beads, adsorbents, nitrocellulose paper, etc.). In order to screen large libraries of test compounds (e.g., drugs, new antimicrobials, etc.), the screening assays of the present invention are preferably conducted in a microplate format.
The present invention contemplates a variety of assay formats. In one embodiment, said first protein (encoded by an oligonucleotide comprising at least a portion of the zipA gene) is immobilized. In another embodiment, said second protein (capable of binding to said first protein) is imrnobilized. In one embodiment, said second protein is FtsZ. In one embodiment, said second protein is labelled (e.g., radiolabelled).
It is not intended that the invention be limited by the means or method of detection. For example, the detection means might be a plate reader, a scintillation counter, a mass spectrometer or fluorometer.
It is not intended that the invention be limited by the nature of test compounds. Such compounds may be synthetic compounds or naturally available compounds.
The method of the present invention is particularly useful for identifying antimicrobials effective against bacteria. However, such identified drugs may have activity against other single cell and multicellular organisms, including, but not limited to fungi, mycoplasma and protozoa.